With a rapidly aging population, diseases of the vascular system are of great concern to our society. Arterial thrombosis is the major cause of death in the form of heart attacks and strokes, while venous thrombosis is associated with pulmonary embolism which occurs after surgery or extended periods of inactivity.
Thrombin is a multifunctional serine protease whose role in thrombosis and hemostasis has been documented by a number of sources (See generally, Tapparelli, et al. TiPS 1993, 14, 366-76). Thrombin acts as a procoagulant through proteolytic cleavage of fibrinogen to form fibrin and as an anticoagulant through activation of the protein C pathway (followed by inactivation of coagulation factors V and VIII.) The concentration of active thrombin is limited by a number of feedback mechanisms involving endogenous factors and proteins. In addition to protein C, antithrombin III is another regulating protein which forms a complex with endogenous heparin. This complex binds to active thrombin, thus inactivating it.
Current anticoagulant therapy consists of three classes of compounds: heparins, coumarins and low molecular weight heparins. These drugs act indirectly to limit the concentration of active thrombin. Heparins and low molecular weight heparins interact with antithrombin III and the coumarins inhibit a number of vitamin K dependent coagulation factors. Although these drugs are prescribed for diseases associated with venous thrombosis and arterial thrombosis, their use is limited. They have a number of side effects, a slow onset of action and only the coumarins are orally active (warfarin and dicumarol).
Indirect thrombin inhibitors have been shown to be less effective at controlling associated diseases than direct thrombin inhibitors. Thus the search for orally active direct thrombin inhibitors is underway in a number of laboratories. These efforts have produced a number of acyclic peptidyl compounds which directly inhibit thrombin. PPACK, argatroban, (D)-NAPAP, hirulog-1 and DUP 714 are examples of these inhibitors . Many of these compounds lack useful oral activity, and many have a poor selectivity for thrombin versus other serine proteases. Therefore, a need remains for new direct thrombin inhibitors.
When compared to acyclic peptides, cyclic peptides have a number of structural features that have been linked to changes in the biological activity of simple peptides. Due to the absence of polar end groups and a relatively rigid structure, cyclic peptides are hypothesized to be more membrane permeable and less susceptible to peptidases. Potentially one could incorporate the structure of simple peptides, within rigid, non-polar macrocyclic framework, to produce active bioavailable compounds.
Cyclotheonamide A (CtA) is a cyclic peptide which was isolated from Theonella.sp, a marine sponge. It inhibits a variety of serine proteases particularly thrombin and trypsin. ##STR3##
Although this molecule inhibits thrombin (Ki# ca. 1-2 nM), it is a scarce natural product which is difficult to extract from its natural source. In addition, CtA is not an optimal candidate for treating thrombin-related disorders as its selectivity for thrombin over trypsin does not favor thrombin. The invention described below claims a novel macrocyclic peptides that inhibit thrombin at nanomolar levels and exhibit reasonable selectivity for thrombin over trypsin.
The invention relates to new compounds of the Formula I ##STR4## wherein: m is 2to 12;
A is ##STR5## where the amido carbonyl is bound to B and the .alpha. aminomethine is bound to the depicted ring carbonyl, PA1 B is ##STR6## where the amido carbonyl of B is bound to the depicted ring methylenes and the methine is bound to A, PA1 R.sub.5 and R.sub.6 are each hydrogen or taken together with the carbon to which each is attached to form a carbonyl, and PA1 b is 0 or 1; PA1 G is ##STR7## where the amine of G is bound to the ring methylenes and the methine is bound to the depicted amide, PA1 R.sub.7 is independently selected from the group consisting of hydrogen, C.sub.1-5 alkyl, carboxyC.sub.1-5 alkyl, phenyl, substituted phenyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), phenylC.sub.1-5 alkyl, substituted phenylC.sub.1-5 alkyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), 3-pyridylC.sub.1-5 alkyl, 4-pyridylC.sub.1-5 alkyl, diphenylC.sub.1-2 alkyl, and naphthyl, substituted naphthyl (where the naphthyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), PA1 E is carbon or C(CH.sub.2).sub.q --, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 24, PA1 R.sub.8 and R.sub.9 are hydrogen or taken together with the carbon of E to form a carbonyl, and PA1 g is 0 or 1; PA1 and pharmaceutically acceptable salts thereof. PA1 R.sub.2 is hydrogen; PA1 m is 2to 12; PA1 A is ##STR9## where the amido carbonyl is bound to B and the a aminomethine is bound to the depicted ring carbonyl, PA1 R.sub.3 is hydrogen or C.sub.1-5 alkoxy, PA1 n is 1 or 2, and PA1 a is 0 or 1; PA1 B is ##STR10## where the amido carbonyl of B is bound to the depicted ring methylenes and the methine is bound to A, PA1 R.sub.4 is selected from the group consisting of hydrogen, C.sub.1-5 alkyl, carboxyC.sub.1-5 alkyl, phenyl, substituted phenyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), phenylC.sub.1-5 alkyl, substituted phenylC.sub.1-5 alkyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), 3-pyridylC.sub.1-5 alkyl, 4-pyridylC.sub.1-5 alkyl, diphenylC.sub.1-2 alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine, bromine or chlorine), PA1 R.sub.5 and R.sub.6 are each hydrogen or taken together with the carbon to which each is attached to form a carbonyl, PA1 b is 0 or 1; PA1 G is ##STR11## where the amine of G is bound to the ring methylenes and the methine is bound to the depicted amide, PA1 R.sub.7 is selected from the group consisting of hydrogen, C.sub.1-5 alkyl, carboxyC.sub.1-5 alkyl, phenyl, substituted phenyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), phenylC.sub.1-5 alkyl, substituted phenylC.sub.1-5 alkyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), 3-pyridylC.sub.1-5 alkyl, 4-pyridylC.sub.1-5 alkyl, diphenylC.sub.1-2 alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine, bromine or chlorine), PA1 E is carbon or C(CH.sub.2).sub.q --, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 25, PA1 R.sub.8 and R.sub.9 are each hydrogen or taken together with the carbon of E to form a carbonyl, PA1 g is 0 or 1; PA1 or pharmaceutically acceptable salts thereof. PA1 W is nitrogen, sulfur or oxygen; PA1 A is ##STR13## where the amido carbonyl is bound to B and the .alpha. aminomethine is bound to the depicted ring carbonyl, PA1 R.sub.3 is hydrogen, hydroxy or C.sub.1-5 alkoxy, PA1 n is 1 or 2; PA1 a is 0 or 1; PA1 B is ##STR14## where the amido carbonyl of B is bound to the depicted ring methylenes and the methine is bound to A, PA1 R.sub.4 is selected from the group consisting of hydrogen, C.sub.1-5 alkyl, carboxyC.sub.1-5 alkyl, phenyl, substituted phenyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), phenylC.sub.1-5 alkyl, substituted phenylC.sub.1-5 alkyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), 3-pyridylC.sub.1-5 alkyl, 4-pyridylC.sub.1-5 alkyl, diphenylC.sub.1-2 alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), PA1 R.sub.5 and R.sub.6 are each hydrogen or taken together with the carbon to which they are attached to form a carbonyl, PA1 b is 0 or 1; PA1 G is ##STR15## where the amine of G is bound to the ring methylenes and the methine is bound to the depicted amide, PA1 R.sub.7 is independently selected from the group consisting of hydrogen, C.sub.1-5 alkyl, carboxyC.sub.1-5 alkyl, phenyl, substituted phenyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), phenylC.sub.1-5 alkyl, substituted phenylC.sub.1-5 alkyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), 3-pyridylC.sub.1-5 alkyl, 4-pyridylC.sub.1-5 alkyl, diphenylC.sub.1-2 alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), PA1 E is carbon or C(CH.sub.2).sub.q --, where q is 0 to 12, with the proviso that the sum of q and m cannot exceed 25, PA1 R.sub.8 and R.sub.9 are each hydrogen or taken together with the carbon of E to form a carbonyl, PA1 g is 0 or 1; PA1 or pharmaceutically acceptable salts thereof. PA1 A is ##STR16## where R.sub.3 is hydrogen and n is 1; B is ##STR17## where R.sub.4 is 2-methyl-1-propyl and R.sub.5 is taken together with R.sub.6 to form a carbonyl. A known N-protected .alpha.-amino acid Ia, is coupled at room temperature to a known C-protected amino acid Ib, using HOBT/DCC in an inert solvent, such as DMF, CH.sub.3 CN or THF, over 5-24 h. Although HOBT/DCC is the preferred coupling agent other agents can be used and include: BOP, BOP-Cl and PyBrOP. The protecting groups are chosen in order to permit selective removal, where the favored protecting groups are CBZ for nitrogen and t-butoxycarbonyl for carboxy. However, other well known protecting groups may be substituted and are described in Green, Theodora Protecting Groups in Organic Synthesis; John Wiley & Sons, New York, 1981. As illustrated the CBZ group is removed by hydrogenation at approximately 20 psig using Pd(OH).sub.2 /C as a catalyst. However, other conditions may be used such as catalytic transfer hydrogenation using Pd/C and formic acid. The resulting C-protected di-peptide Ic, is coupled to an N-protected aliphatic amino acid, Id, followed by removal of the N-protecting group to give amine Ie. As illustrated, the CBZ serves as the N-protecting group and Pd/(OH).sub.2 is the hydrogenation catalyst. However either the protecting group or the reaction conditions may be modified as previously described. Intermediate Ie is coupled to 6-imino4-methylbenzenesulfonyl)-amino!methyl!amino!-2-(R,S)-2-(trimet hylsilyl)ethoxy!methoxy!-3(S)-9-phenylmethoxycarbonyl)-amino!hexanoic acid, ( Maryanoff et al. Journal of the American Chemical Society 1995, 117, 1225-39) using HOBT/DCC at room temperature for 4-24 h in an inert solvent and deprotected with Pd(OH).sub.2 to give the arginine derivative If. The t-butoxycarbonyl and SEM protecting groups are removed with TFA and the resulting intermediate is coupled at room temperature with BOP-Cl and DMAP in an inert solvent such as CH.sub.2 Cl.sub.2 to give the hydroxy macrocyclic derivative Ig. Compound Ig is oxidized using the Dess-Martin periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger such as anisole, thioanisole, pentamethylbenzene, dimethylsulfide or cresol to give a compound of Formula I. PA1 A is ##STR23## where R.sub.3 is hydrogen and n is 1; B is ##STR24## where R.sub.4 is benzyl and R.sub.5 is taken together with R.sub.6 to form a carbonyl; and PA1 G is ##STR25## where E, R.sub.8 and R.sub.9 are taken together to form a carbonyl and R.sub.7 is 4-chlorobenzyl. PA1 A is ##STR33## where R.sub.3 is hydrogen and n is 2; B is ##STR34## where R.sub.4 is benzyl and both R.sub.5 as well as R.sub.6 are hydrogen. A known N-protected aldehyde IIIa, is reductively aminated at room temperature to a known C-protected amino acid IIIb, using NaB(OAc).sub.3 H in an inert solvent, such as CH.sub.2 Cl.sub.2 or (CH.sub.2).sub.2 Cl.sub.2 , over 2-16 h. The protecting groups are chosen in order to permit selective removal, where the favored protecting groups are Fmoc for nitrogen and t-butoxycarbonyl for carboxy. However, other protecting groups may be substituted as previously discussed. The free amine of the resulting product IIIc is protected as the CBZ and the Fmoc group of the other amine is cleaved with an anhydrous base such as piperidine to give IIId. Intermediate IIId is coupled to 6-imino4-methylbenzenesulfonyl)-amino!methyl!amino!-2-(R,S)-2-(trimet hylsilyl)ethoxy!methoxy!-3(S)-9-fluorenylmethoxycarbonyl)-amino!hexanoic acid, (Maryanoff et al. Journal of the American Chemical Society 1995, 117, 1225-39) using HOBT/DCC at room temperature for 4-24 h in an inert solvent and deprotected with Pd(OH).sub.2 to give the arginine derivative IIIe. The Fmoc group is removed with an organic base and the t-butoxycarbonyl and SEM protecting groups are removed with TFA. The resulting intermediate is coupled at room temperature with BOP-Cl and DMAP in an inert solvent such as CH.sub.2 Cl.sub.2 to give the hydroxy macrocyclic derivative IIIf. Compound IIIf is oxidized using the Dess-Martin periodinane in an anhydrous aprotic solvent and deprotected using HF in the presence of a carbocation scavenger to give a compound of Formula I. PA1 A is ##STR40## where R.sub.3 is hydrogen and n is 1; and ##STR41## G is where E, R.sub.8 and R.sub.9 are taken together to form a carbonyl and R.sub.7 is phenyl. PA1 A is ##STR47## where R.sub.3 is methoxy; n is 1; PA1 B is ##STR48## where R.sub.4 is 4-chlorobenzyl and R.sub.5 is taken together with R6 to form a carbonyl; and PA1 G is ##STR49## where E is carbon, R.sub.8 and R.sub.9 are hydrogen and R.sub.7 is hydrogen. PA1 A is ##STR61## where R.sub.3 is hydrogen and n is 1; B is ##STR62## where R.sub.4 is benzyl and R.sub.5 is taken together with R.sub.6 to form a carbonyl; and PA1 G is ##STR63## where E, R.sub.8 and R.sub.9 are taken together to form a carbonyl and R.sub.7 is benzyl.
R.sub.3 is hydrogen, hydroxy or C.sub.1-5 alkoxy, PA2 n is 1 or 2 and PA2 a is 0 or 1; PA2 R.sub.4 is selected from the group consisting of any of hydrogen, C.sub.1-5 alkyl, carboxy C.sub.1-5 alkyl, phenyl, substituted phenyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine, bromine or chlorine), phenylC.sub.1-5 alkyl, substituted phenylC.sub.1-5 alkyl (where the phenyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine), 3-pyridylC.sub.1-5 alkyl, 4-pyridylC.sub.1-5 alkyl, diphenylC.sub.1-2 alkyl, naphthyl or substituted naphthyl (where the naphthyl substituents are C.sub.1-5 alkyl, carboxy C.sub.1-5 alkoxycarbonyl, carboxamido, amino, C.sub.1-5 alkylamino, hydroxy, C.sub.1-5 alkylcarbonylamino, C.sub.1-5 alkoxy, fluorine bromine or chlorine),
An additional aspect of the invention relates to novel compounds of the Formula II which are intermediates in the synthesis of compounds for the Formula I, ##STR8## wherein: R.sub.1 is hydroxy;
Yet another aspect of the invention relates to novel thrombin inhibitors of the Formula III. ##STR12## wherein: m is 2 to 12;
The terms used in describing the invention are commonly used and known to those skilled in the art. However, the terms that could have other meanings are defined. "Independently" means that when there are more than one substituent, the substitutents may be the same or different. The term "alkyl" refers to straight, cyclic and branched-chain alkyl groups and "alkoxy" refers to O-alkyl where alkyl is as defined supra. "CBZ" refers to benzyloxycarbonyl. "BOC" refers to t-butoxycarbonyl and "Ts" refers to toluenesulfonyl. "DCC" refers to 1,3-dicyclohexylcarbodiimide, "DMAP" refers to 4-N'N-dimethylaminopyridine and "HOBT" refers to 1-hydroxybenzotriazole hydrate. "FMoc" refers to N-(9-fluorenylmethoxycarbonyl). Amino acid refers to compounds where the amino group and the carboxy group are on different carbon atoms. The term .alpha.-amino acid, refers to compounds where both the carboxy and the amino group are attached to the same carbon atom. The stereochemistry of this carbon is indicated by the terms "D and L" where D indicates right-handed chirality.